The hormone-directed, cyclic AMP-mediated phosphorylation and dephosphorylation of enzymes and regulatory proteins are critical in the control of myocardial contractility, glycogen metabolism and many other cellular processes. The dephosphorylation reaction catalyzed by protein phosphatases is poorly understood. This applicant has isolated and identified five protein phosphatases siozyme species from canine cardiac muscle. Among these, three nonspecific, divalent cation-independent isozymes with molecular weights ranging from 95-161,000 appear to consist of a common catalytic subunit of Mr equals 35,000 and an inhibitory regulatory subunit(s). The other two isozymes (Mr equals 49,300 and 61,000) are divalent cation-dependent and exhibit a narrow substrate specificity. The catalytic subunit of Mr equals 35,000 has been purified to apparent homogeneity. Purification of the nonspecific, divalent cation-independent isozymes to homogeneity will be first carried out. The homogeneous preparation will be submitted to characterization with respect to their subunit composition, substrate specificity, physical, kinetic and regulatory properties. Experiments will be carried out to search for the postulated inhibitory regulatory subunit(s) and the unknown second messenger involved in the regulation of the holoenzymes. Characterization of the purified catalytic subunit of Mr-35,000 with respect to physical, chemical and kinetic properties, functional group(s) in active site, amino acid composition, metal content, mechanism of regulation and mechanism of dephosphorylation will be carried out. Sarcoplasmic reticulum (SR)-bound protein phosphatase will be studied in parallel with the cytoplasmic enzyme. The regulatory role of protein phosphatases in SR Ca 2 ion-uptake and glycogen metabolism will be elucidated.